Research focuses on neuroendocrine mechanisms of adaptation to stress, with emphasis on the regulation of the hypothalamic pituitary adrenal (HPA) axis. Previous studies of this laboratory demonstrated that repeated stress is associated with either desensitized or maintained ACTH responses to an homotypic stimulus but invariable enhanced responses to a heterotypical stress. Extension of these studies showed similar facilitation of the HPA axis activity 10 days after a single stress exposure. To study the mechanism of these responses, in situ hybridization studies with intronic probes were used to study CRH and VP transcription in the PVN. It was shown that preservation of ACTH responses to an homotypic repeated stimulus correlates with increased CRH expression, whereas hyperresponsiveness to a heterotypical superimposed stress correlates with increased VP expression. The increase in CRH in PVN neurons precedes that of VP, and is self-limited independently of the levels of circulating glucocorticoid. VP transcription is markedly inhibited by glucocorticoids, but CRH transcription is relatively insensitive to glucocorticoid feedback. While the sensitivity of the onset of CRH and VP responses to stress depends on the prevailing levls of glucocorticoids, the increases of VP but not CRH following the stimulus are dampened by the glucocorticoid surge. The molecular mechanism of the differential regulation of CRH and VP is under current investigation. The lack of correlation between the number of CRH receptors (CRHR) and CRHR mRNA levels suggests the involvement of translational mechanisms on CRHR regulation. The possibility that a short open reading frame (ORF) present in the 5UTR of the CRHR mRNA influences mRNA translation was studied using constructs of luciferase or CRHR main ORF and the native or mutated CRHR-5UTR. Transfection of constructs with upstream ATG mutation were more effective that constructs with the native 5UTR in inducing luciferase activity or CRH binding after transfection into cells suggesting that the upstream ORF in the CRHR inhibits protein expression and that the 5UTR has a role regulating the number of CRHR.Cloning and characterization of the 5flanking region of the V1b VP receptor gene revealed that the gene contains 3 exons, with the first intron located in the 5UTR being required for expression in transfected cells. The putative promoter shows no TATA box but several elements likely to mediate transcription regulation. Current studies focus on the identification of elements responsible for promoter activity and tissue specific expression. - hypothalamic pituitary adrenal (HPA) axis, intronic probes, VP transcription, glucocorticoids, CRH receptors, open reading frame (ORF)